The proposed project asks fundamental and applied questions concerning the role that Mycoplasma pneumoniae (Mp) and its newly discovered ADP-ribosylating, vacuolating toxin, designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin, play in the pathogenesis of airway disorders. CARDS toxin remarkably recapitulates the pro-inflammatory cytokine/chemokine profiles and histopathology that accompany Mp infection. In addition, inactivation of CARDS toxin significantly reduced the release of mature form interleukin-1 (IL-1) during Mp infection. IL-1 is a critical pro-inflammatory cytokine that dictates severity of inflammation associated with a wide spectrum of inflammatory diseases. Release of active mature IL-1 is accomplished by inflammasome complex-mediated activation of caspase-1, the enzyme responsible for cleaving immature pro-IL-1 into its mature form. NLRP3 is a key component of the inflammasome complex, and multiple signals and stimuli trigger formation of the NLRP3 inflammasome complex. In the airway, Mp and CARDS toxin are primarily detected in the alveolar macrophages, and our preliminary studies show NLRP3 inflammasome activation by CARDS toxin in primary macrophages leading to IL-1 production. CARDS toxin co-localized with NLRP3 inflammasome in primary macrophages and interacted with NLRP3 as deduced by co-immunopreciptation analysis. Surprisingly, our preliminary studies demonstrated CARDS toxin-mediated ADP-ribosylation of NLRP3 and a requirement of ADP- ribosyltransferase (ART) activity of CARDS toxin for NLRP3 activation. Based on our preliminary results, we hypothesize that Mp and specifically CARDS toxin are key mediators of airway dysfunction and inflammation via CARDS toxin ART-related mechanisms involving NLRP3 inflammasome activation and resulting release of active mature IL-1 in the airway. We plan to test this hypothesis by - a) studying the role of CARDS toxin ADP ribosylation and binding activities in NLRP3 inflammasome assembly and IL-1 secretion, b) examining in vivo involvement of NLRP3 in CARDS toxin mediated IL-1 production, c) investigating the role of IL- in triggering CARDS toxin-mediated lung inflammatory disease pathology, and d) elucidating the role of ART activity of CARDS toxin during in vivo IL-1 production and inflammasome activation. Our long-term goal is to develop effective strategies to diagnose, treat and prevent Mp-related airway diseases in a substantial population of both children and adults. Understanding the mechanisms by which the inflammasome is activated via CARDS toxin should lead to therapeutic interventions and improved health in many individuals who suffer from acute and chronic airway and extrapulmonary pathologies linked to Mp infections. The proposed studies will also provide new insights about inflammasome activation mechanisms since post- translational ADP-ribosyltransferase-mediated modification of the inflammasome is a novel mechanism for inflammasome activation with subsequent release of IL-1 and associated pathologies.